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Staphylococcus aureus has the capacity to grow anaerobically in the presence of glucose without a functional electron transport system or without a complete tricarboxylic acid cycle.1 In the presence of oxygen, a membrane-bound electron transport system is formed and the growth rate increases significantly.2 When grown aerobically on nutrient agar where the principal carbon sources are amino acids, mutants that form the electron transport system very slowly can easily be differentiated from the wild type by the difference in colony size. The synthesis of protoheme (Fe protoporphyrin IX) is required for the formation of the heme enzymes of the electron transport system. Although the genetic analysis of the heme biosynthetic chain has been long sought in bacteria, it was not until the fortunate use of kanamycin on Staphylococcus that mutants in the protoheme biosynthetic chain could be readily prepared and tested for gene sequence by phage transduction. Some of the mutants are able to grow as rapidly as the wild type when 8 ,uM protoheme is added to the nutrient agar. The preparation of these mutants and their analysis by transduction is reported in this study. Methods.-Strain U-71 of S. aureus and International Staphylococcal Typing phage 53 were supplied by J. W. Baldwin, University of Georgia. Bacteria were grown in nutrient agar or broth and trypticase soy agar or broth (Difco Products Corp.) at 370C. The protoheme precursors: 5 aminolevulinic acid (ALA), protoporphyrin, protoheme (Sigma Chemical Corp.), or porphobilinogen (PBG) (the gift of D. C. Mauzerall, Rockefeller University) were added at 8 uM where indicated in the text. Protoheme or protoporphyrin (20 mg) was dissolved in 0.5 ml pyridine, diluted with 0.5 M NH40H to 5 ml, and stored in the dark at 16°C. To select the mutants, a suspension of 107 cells from an 18-hr culture was plated on nutrient agar containing 6 ,uM kanamycin sulfate (Bristol Labs.). The plates were incubated 48 hr and the minute colonies transferred to protoheme-containing plates with sterile hardwood toothpicks. Mutants able to grow as rapidly as the wild type in the presence of protoheme were collected. Double mutants were selected by growing ALA auxotrophs with kanamycin and harvesting the small colonies from a nutrient agar plate containing ALA. The small-colony mutants from plates containing ALA, which grew normally in the presence of protoheme, were shown by transduction to have two lesions in the protoheme biosynthetic pathway. Transduction and phage propagation techniques were essentially those described by Pattee and Baldwin.3 Briefly, the recipient culture was grown on nutrient agar with protoheme for 18 hr and suspended at a density of 1010 cells/ml in the presence of 1.5-2.0 X 1010 phage/ml for 30 min at 37°C. The mixture was then placed in an ice bath, made to 11.3 mM with sodium citrate, and centrifuged. The pellet was resuspended in cold 67 mM citrate and a tenth of the volume plated on nutrient agar containing 0.19 mM sodium citrate. Controls for back mutation and phage sterility were included in each experiment. Bacteria for phage propagation were grown on trypticase soy agar for 12 hr anid harvested. The bacteria were combined with 1010 phage in trypticase soy broth containing 0.5% agar and 4 mM CaCl2 and poured on trypticase soy agar plates. The plates were incubated for 5 hr, the soft agar was scraped off, and washing was performed with three por-

Linear sequential arrangement of genes for the biosynthetic pathway of protoheme in Staphylococcus aureus.